A genetic approach is described to functionally characterize genes expressed in embryonal stem (ES) cells that are repressed upon differentiation. A promoter trap retrovirus will be used to induce functional beta-galactosidase (lacZ) gene fusions in totipotent ES cells. Clones expressing regulated fusion genes will be identified and cDNAs corresponding to normal, unoccupied alleles will be cloned and sequenced. Transgenic mice containing LacZ fusion genes will be constructed in order to assess the biological functions of disrupted genes and to study the expression of LacZ fusion genes during development. Finally, the biochemical functions of the disrupted genes will be studied in ES cells line established from nullizygous blastulae. Genes repressed as ES differentiate are expected to encode functions required for stem cell functions, including responses to external stimuli, cell-cell interactions, cell proliferation and totipotency. Gene interactions will also be studied. For example, loss of one developmentally regulated gene may alter the expression of other developmentally regulated genes. Genes affecting similar developmental phenotypes may also interact at a biochemical level. These studies may identify genes that are expressed specifically in proliferating cells, including stem cells responsible for tissue renewal in adult animals, and may be involved in malignant transformation. The utility of this approach has been demonstrated. 2 fusion genes have been identified that are tightly repressed during differentiation, including one involving the developmentally regulated REX-1 gene. The REX-1 protein contains 4 zinc finger domains and a highly acidic region, suggesting a possible function as a transcription factor. 9 of 12 fusion genes tested have been successfully transferred into the germline of mice, including the disrupted REX-1 locus.